Bacterial toxoids and gram-negative immune globulin therefrom

ABSTRACT

ADP-ribosylating toxins are rendered exzymatically inactive by reactions with photolabile affinity reagents. The toxoids retain the antigenic and immunogenic properties of the original toxins. These bacterial toxoids can be used as immunogens to protect against the specific disease that the precursor toxins cause or, in the case of P. aeruginosa, the toxiod can be used in combination with E. coli J-5 vaccine to protect against gram-negative bacteremia in general.

BACKGROUND

Inactivation of ADP-ribosylating toxins has been attempted by usingcross-linking agents such as formaldehyde and glutaraldehyde. Cryz etal., "Effect of Formalin Toxoiding on Pseudomonas aeruginosa Toxin A:Biological, Chemical, and Immunochemical Studies," Infec. and Immun.,32, No. 2, 759-768, May 1981 describe such a toxoid. Two problems withsome such toxoids have been reversion to the toxic form and loss ofantigenicity and immunogenicity. The possibility of their reverting tothe toxic state effectively precludes ever obtaining human antisera fromsuch toxoids or their use as active vaccines.

SUMMARY OF THE INVENTION

It has now been found that ADP-ribosylating toxins such as exotoxin Afrom P. aeruginosa can be converted into toxoids with certainphotolabile affinity reagents. The process is irreversible and thetoxoid retains the antigenic and immunogenic properties of the parenttoxin. Thus, the toxoid is useful as an immunogen against the specificdisease caused by the parent toxin, or, in the case of P. aeruginosa,the toxoid can be combined with vaccines to bacterial endotoxins orantisera produced therefrom to offer a broader spectrum of protectionagainst gram-negative bacteremia than heretofore possible.

An object of this invention is bacterial toxoids derived fromADP-ribosylating toxins, which are antigenic and immunogenic, and whichdo not spontaneously revert to the toxic state.

Another object is a process for producing such toxoids by reacting thecorresponding toxins with certain photolabile affinity reagents.

A further object of this invention is the combination of the specifictoxoid from P. aeruginosa with certain endotoxin vaccines. The vaccinescould be used to induce antisera in mammals. The immune globulinobtained therefrom could be used to therapeutically treat acutegram-negative sepsis or used prophylactically and, in the case of P.aeruginosa bacteremia, to help prevent or cure this most devastatingform of sepsis. Alternatively, the vaccines could be used to preventthese infections by active immunization.

DETAILED DESCRIPTION OF THE INVENTION

The ADP-ribosylating toxins which can be used in this invention arethose which exhibit what is known in the art as ADP-ribosyltransferaseactivity and NAD-glycohydrolase activity. These include the exotoxin-Afrom Pseudomonas aeruginosa, the heat labile (LT) enterotoxin from E.coli, the cholera enterotoxin from Vibrio cholerae, and, in the case ofgram-positive bacteria, the diphtheria exotoxin from Corynebacteriumdiphtheriae. All these toxins are known.

P. aeruginosa produces not only an endotoxin which is common togram-negative bacteria, but also an exotoxin referred to as exotoxin-A.The exotoxin is a protein (mw approx. 70,000) which functionsintracellularly as the enzyme with substrate specificity for NAD andcauses its toxic effect by the ADP-ribosylation of Elongation Factor 2,thereby irreversibly preventing protein synthesis in the target cell.The toxoid of this invention has greatly reduced enzymatic activity(i.e., it is non-toxic) but is highly antigenic and immunogenic. Thematerial is used to raise antibodies in mammals. The resulting immuneplasma is used either as a monovalent or, in combination with immuneplasma (or isolated antibodies therefrom) raised against genericantigens to endotoxin, as a divalent immuno-therapeutic or prophylacticfor gram-negative sepsis.

It is within the scope of this invention to enhance the immune responseof the toxoids by binding said toxiods to a protein such as keyholelimpet hemocyanin.

In the process of this invention, the exotoxin is mixed with thephotolabile affinity reagent, preferably in aqueous solution, in molarratios ranging from 1:100 to 1:10,000. A ratio of 1:1300 has been foundto work well. Degradation of the exotoxin and toxoid is minimized bymaintaining the reagent vessel at about 0° C. and maintaining an inertatmosphere (e.g., N₂) in the vessel. The mixture is exposed to aneffective amount of non-denaturing light, i.e., light containing aninsubstantial amount of the U.V. portion of the spectrum. Afteractivation by the light, the reacted material is purified, as by passagethrough an elution column.

The preferred photolabile affinity reagent is 8-azidoadenosine. Anotherwithin the scope of this invention is 8-azidoadenine, which has beenfound to be moderately effective. In the process of this invention, the8-azidoadenine or 8-azidoadenosine, when irradiated, loses nitrogen inthe form of N₂ and forms unstable nitrene intermediates (I). Saidnitrenes then combine with the ADP-ribosylating toxins to form the noveltoxoids: 8-adenylamino toxin and 8-adenosylamino toxin, respectively(II): ##STR1## wherein RH is the ADP-ribosylating toxin. The complexescan, therefore, be described structurally as: ##STR2## wherein R is anADP-ribosylating toxin radical. Thus, a toxoid of this invention derivedfrom P. aeruginosa exotoxin A and 8-azidoadenine is 8-adenylamino P.aeruginosa exotoxin A; from 8-azidoadenosine it is 8-adenosylamino P.aeruginosa exotoxin A.

The invention is further defined by reference to the followingpreparations and examples, which are intended to be illustrative and notlimiting.

The following procedure can be used to prepare, isolate, and purify P.aeruginosa exotoxin A.

PREPARATION 1 Purified Exotoxin A of P. Aeruginosa Step 1: DE-52Preparation

a. Two batches of excess DE-52 (Whatman DE-52 diethylaminoethylcellulose, pre-swollen) (600-700 ml dry volume) were suspended in twicethe volume of water and allowed to settle. The water was poured off andthe DE-52 resuspended. With the second settling, a significant reductionin fines was noticed in the water. If this was not observed, it wasrepeated.

b. The DE-52 batches were washed over coarse fritted glass filters with3200 ml of 0.5 N NaOH followed by 3200 ml H₂ O, 3200 ml 0.5 N HCl, 3200ml H₂ O, 3200 ml 0.5 N NaOH and H₂ O until the pH was below 8.

c. The DE-52 was washed with several volumes of 0.01 M NaCl 0.01 M Trisbuffer, pH 8.0.

d. 100 ml of washed DE-52 was used to prepare a K 16/40 column, whichwas run at room temperature with a flow rate of 1.4 ml/min. The columnwas washed with 0.05 M NaCl, 0.01 M Tris pH 8.0 until it wasequilibrated (1 to 2 days).

Step 2: Sephadex G-75 Column Preparation

a. 136 g G-75 (Pharmacia) was rehydrated in 3 L 0.5 M NaCl, 0.1 M Tris,pH 8.0, boiled to degas and then cooled to 4° C. overnight.

b. A Pharmacia K 50/100 column was packed with the G-75 (ca. 6 h) at 4°C. and washed with buffer containing 0.02% w/v sodium azide.

c. The column was checked with 40 mg blue dextran 2000 (Pharmacia) in 20ml buffer at a flow rate of 0.5 ml/min collecting 7.6 ml fractions at 37cm head pressure. The V_(o) was 714 ml (40% of the bed volume) and thedilution factor was 5.3. The column was maintained at 4° C.

Step 3: Hydroxylappaltite (HTP) Column Preparation

a. 120-150 ml dry volume of Hydroxylappaltite, (BIO-GEL HTP, Bio-Rad No.130-0420) was suspended in twice the volume of 0.005 M NaH₂ PO₄, 0.1 MNaCl pH 8.0 buffer.

b. A Pharmacia K 26/40 column was packed with the HTP at a flow rate of1.5 ml/min. at 4° C. and washed with buffer.

Step 4: Fermentation Media--TSB-D (CM 679)

a. Trypticase soy broth (Baltimore Biological Laboratory (BBL) No.11768) was deferrated by mixing 1800 g TSB with 600 g Chelex 100, minus400 mesh sodium form (Bio-Rad No. 142-2852), in 5400 ml water at roomtemperature for 5 to 6 hours (10×concentrate). It was then filteredthrough Whatman No. 1 filter paper and frozen until used.

b. The chelated medium (a above) was diafiltered through an H10P10hollow fiber cartridge in an Amicon Corp. DC-30 system. The concentratewas diluted in distilled water to 1×(60 L) and diafiltered to yield 40 LTSB-D. The medium had a pH of 7.0 to 7.5; a chloride concentration ofapproximately 0.1 M, and was sterilized by filtration (0.45 μm). Themedium was stored at 4° C.

c. On the day prior to fermentation, 2 L of TSB-D was asepticallyremoved. To 1600 ml, 374 g mono-sodium glutamate and 400 ml glycerin wasadded, mixed at room temperature until dissolved, filtered through 0.45μm filters and added as enriched medium to the fermentation vesselyielding a final concentration of 0.05 M monosodium glutamate and 1.0%glycerin. The remaining 400 ml of non-enriched medium was saved for theseed flasks.

Tryptic Soy Agar Slants

a. 40 g of TSA was allowed to swell in 1 L water for 15 min. The mixturewas boiled for one minute, aliquoted (8 ml per 1.2×12 cm screw-cappedtubes) autoclaved 15 min, 121° C., 15 psi, then placed at a 15° angle tomake long slants.

Growth

a. One loopful each of the frozen stock of P. aeruginosa strain PA-103(ATCC 29260)* was streaked onto four TSA (Tryptic soy agar, Difco)slants and incubated at 37° C. for 18 to 20 hours.

b. The growth from all four slants was washed off with a total of 4 mlof medium. One ml of the suspension was used to inoculate each of four250 ml baffled flasks containing 100 ml of non-enriched TSB-D. Thecultures were incubated with shaking at 32±1° C. and 250 oscillationsper minute for 5 to 6 hours.

c. All 400 ml of seed was used to inoculate 40 L of enriched TSB-D. Thefermentation medium was saturated with O₂. Fermentation proceeded at32±1° C. with stirring while monitoring the pH (range 7.0-8.0) andoptical density (to stationary phase growth) and maintaining thedissolved oxygen level at less than 25% of saturation for 18-24 hours.

d. The bacteria were removed by centrifugation (20,000 rpm) in a modelKII centrifuge (Electro-Neucleonics) and the supernatant fluidpre-filtered and filter sterilized (0.45 μm).

Step 5: Concentration

a. Using an Amicon DC-10 hollow fiber system with an H10P10 cartridgethe volume of supernatant fluid was reduced from 40 L to 3 to 4 L bydiafiltration).

b. Cold water was added to the retentate to bring the level to 15 L andthe pH was determined to be within 7.0 to 7.5 and the chlorideconcentration to be 0.02 M. If the Cl⁻ concentration was notsufficiently reduced, the diafiltration was continued until anacceptable concentration was obtained.

Step 6: DE-52 Batch Fractionation

a. 2 L of washed DE-52 was added to the diluted retentate and mixed atroom temperature for about 2 hours maintaining a pH of 8.0. When the pHwas stable, the suspension was cooled to 4° C. and allowed to settleovernight.

b. Approximately 14 to 15 L of supernatant fluid was siphoned off andthe cellulose was poured onto a 2.5 L fritted glass filter. Theremaining fluid was removed under vacuum leaving the cellulose moist.

c. The cellulose was washed by filtration successively with 2.5 L of0.01 M NaCl, 0.01 M Tris pH 8.0; 0.05 M NaCl, 0.01 M Tris pH 8.0; and0.25 M NaCl, 0.01 M Tris pH 8.0. The cellulose was discarded.

d. An enzyme assay, described below, was performed to confirm that thetoxin was in the 0.25 M NaCl fraction before proceeding with furtherfractionation.

e. The 0.25 M NaCl active fraction was precipitated by adding solidammonium sulfate at 70% (4° C.) saturation maintaining a pH of 8. Theactive fraction was held overnight for complete precipitation.

Step 7: G-75 Desalting

a. The precipitated fraction was mixed well and approximately 1/3 of thesuspension was removed, holding the remainder at 4° C.

b. The aliquot was centrifuged at 16,000×g at 4° C. for 20 minutes(Sorvall RC5B, GSA rotor, 10,000 rpm) and the supernatant fluid wasdiscarded.

c. The pellet was gently resuspended in 30 ml of 0.5 M NaCl, 0.1 M Tris,pH 8.0 buffer containing 0.02% Na azide. For clarification it wascentrifuged at 17,000×g for 5 min. 4° C. It was then pre-filtered (0.8μm) and then filter sterilized (0.45 μm).

d. The sample was chromatographed through the G-75 column at a flow rateof 1.5 ml/min and 7.5 ml fractions were collected. The fractions wereassayed for toxin by countercurrent immunoelectrophoresis (Hyland CEPSupply Package, Hyland, Lab) and the toxin-containing fractions werepooled. The immunoelectrophoresis was performed using monospecificequine antiserium to toxin (See Step 9-b).

e. The pool was precipitated with ammonium sulfate at 70% saturation, pH8 and stored at 4° C.

f. Steps (a) to (e) were repeated twice more with the remaining DE-52fraction. The precipitates were then pooled.

Step 8: HTP Fractionation

a. The G-75 precipitates were collected by centrifugation as before.

b. The pellet was gently dissolved in 20 ml 0.005 M NaH₂ PO₄, 0.1 M NaClpH 7.0 starting buffer and dialyzed against 2 L of buffer at 4° C.overnight.

c. The sample was run through an Hydroxylappaltite Bio-Gel HTP Bio-Rad)column at a flow rate of 1.5 ml/min and chased with 3 bed volumes of0.01 M NaH₂ PO₄, 0.1 M NaCl pH 7.0. The optical density (O.D.) at 280 nmwas monitored on a Beckman Spectrophotometer model 26. The non-adsorbingmaterial was completely eluted from the column before proceeding to thenext step.

d. The buffer was changed to 0.06 M NaH₂ PO₄, 0.1 M NaCl pH 7.0 and 7 mlfractions were collected. The O.D. peak was assayed by CIE.

e. The toxin fraction was pooled and precipitated by dialysis againstsaturated ammonium sulfate at 4° C. overnight.

Step 9: DE-52 Gradient Fractionation

a. The precipitate was collected by centrifugation at 12,000×g for 10minutes at 4° C. (Sorvall SS34 rotor, 10,000 rpm). It was then gentlyresuspended in 5 ml 0.05 M NaCl, 0.01 M Tris pH 8.0 (starting buffer)and dialyzed against 1 L of the starting buffer twice at 20° C. for 18to 24 hours.

b. DE-52 chromatography was performed with a linear 1 L gradient of 0.05M to 0.5 M NaCl in 0.01 M Tris pH 8.0. Two and one half ml fractionswere collected at a flow rate of 1.4 ml/min. The toxin peak (O.D.₂₈₀ nm)was eluted at or about 0.15 M Cl⁻ and was assayed by countercurrentimmunoelectrophoresis (CIE) with monospecific equine antiserum at 30 mAfor 60 minutes to determine the fractions to be pooled.

c. A determination was made of the pool O.D.₂₈₀ nm and the proteinconcentration was calculated by the following equation:

    [1% solution O.D..sub.280 nm =18.5].

d. Aliquots of 2.5 ml/6 ml vial were made and stored at -70° C.

The purified exotoxin A was then used to prepare the P. aeruginosatoxoid of Example 1.

EXAMPLE 1 Preparation of P. Aeruginosa Toxoid

12 mg of 8-azidoadenosine (prepared according to Holmes et al., J. Am.Chem. Soc., 87, 1772 (1965)) was covered with 10 ml of pH 7.4 buffer(9.5 mM phosphate, 140 mM NaCl) and shaken until most dissolved, i.e.,forming a saturated solution. The suspension was filtered through asintered glass filter and the concentration of the filtrate determinedby ultraviolet spectroscopy. The filtrate was diluted 1 to 50 with theabove pH 7.4 phosphate buffer and the absorption, λ_(max) 282, ε=14,500determined. The concentration of 8-azidoadenosine was 2.9×10⁻³ M. 7.5 mlof this solution was mixed with 7.5 ml of Pseudomonas aeruginosaexotoxin A (150 μg/ml, 1125 μg total) from Preparation 1. The finalmolar ratio of azido compound to toxin was 1300:1.

This solution was charged (in 2×7.5 ml batches) into a 12 ml Pyrexreactor surrounded by a jacket through which ice water was pumped. Priorto irradiation, the reactor was stoppered with a serum cap through whicha hypodermic needle was inserted, and the vessel was then alternatelyevacuated and flushed with nitrogen. Photolysis was effected by a 450 WHanovia medium pressure lamp (6794 0360) placed 4 inches from thereactor for 6 minutes with cooling.

The solution was then desalted by passage through six Sephadex PD 10columns (addition in 2.5 ml batches and elution with 3.5 ml of pH 7.4buffer). The eluates were assayed by High Performance LiquidChromatography on a 60 cm Toya Soda molecular sieve column. The material(8-adenosylamino P. aeruginosa exotoxin A) was found to be a single peakat about 68,000 Daltons and corresponded to about 55±10 μg/ml asdetermined by peak area (using pure toxin as standard). A similar valuewas determined using a colormetric protein assay (Biorad).

EXAMPLE 2 Toxicity Assays

Exotoxoid prepared as in Example 1 is compared to exotoxin A with regardto cytotoxicity, guinea pig skin reaction, and enzyme activity.

Mouse fibroblasts or L cells from a cell line continuously maintained inour laboratory were used as target cells in an in vitromicrocytotoxicity assay. When these L cells, growing in microculture,are exposed to nanogram amounts of purified exotoxin, they showmicroscopic evidence of cell death (e.g., loss of normal architecture,"balling up," and loss of adherence to plastic surfaces). These visuallyobserved cytotoxic changes correspond to and can be quantified by theinhibition of [³ H] thymidine incorporation by the toxin-exposedcultures. The titer at 50% inhibition is then calculated by graphing thepercent inhibition as a function of the log of the dilution of toxin.

The guinea pig skin test is performed by intradermal injection of 0.1 mlof the test compound at two sites on shaved and depilated adult Hartleywhite guinea pigs. Three days after injection, the areas of reactionsites are measured in mm².

The enzyme assay quantifies the degree of ADP-ribosylation of EF-2 bymeasuring the incorporation of the tracer (adenine-¹⁴ C)-NAD into TCAprecipitable product. Under the assay conditions, the following reactionoccurs: ##STR3## As the presence of exotoxin favors ADP-ribosylation ofEF-2, the amount of radioactivity (counts per minute minus backgroundnoise) in the precipitable product is a measure of the amount of toxinin the sample. If the amount of toxin is held constant, then the amountof radioactivity incorporated into EF-2 is a function of time. Assayingat various time intervals reveals the velocity of the reaction.

Samples of exotoxoid, prepared as in Example 1, and exotoxin A wereassayed as above and the following data were obtained:

                  TABLE 2-1                                                       ______________________________________                                        Cytotoxicity, Skin Reaction, and                                              Enzyme Activity Assays                                                                Cyto.                 Enzyme Velocity                                         ID.sub.50 (ng)/10.sup.3                                                                  Skin React.                                                                              nM ADP-ribose                                   Sample  Cells      Area (mm.sup.2)                                                                          EF-2/min/ng protein                             ______________________________________                                        Toxin    4         153        155                                             Toxoid  887         7          9                                              Reduction                                                                             99%        95%        94%                                             ______________________________________                                    

EXAMPLE 3 Antigenicity Assay

Samples of exotoxin (Preparation 1) and exotoxoid (Example 1) weretested for antigenicity using monospecific equine antiserum. Thefollowing data were obtained.

                  TABLE 3-1                                                       ______________________________________                                        Rocket Immunoelectrophoresis                                                             Peak Hgt. of Rockets (mm)                                                     Avg. of 2 rockets                                                  ______________________________________                                        Toxin (Prep. 1)                                                                            36.5                                                             150 ng                                                                        Toxoid (Ex. 1)                                                                             36.0                                                             150 ng                                                                        ______________________________________                                    

From these data, it is concluded that the toxoid retains approximately100% of the original toxin's antigenicity.

EXAMPLE 4 Potency Assay (Antibody Response)

Mice were vaccinated i.p. on day 1 with 5 μg alum absorbed toxoid(alum-toxoid); on day 7 with 5 μg alum-toxoid i.p.; and on day 21 with 5μg aqueous-toxoid i.v. On day 25, 32 immunized mice were bled andantitoxin titers were determined by ELISA assay.

Sera were diluted 1:40, 1:160, and 1:640. The following definitions wereused in Table 4-1.

    ______________________________________                                                        ELISA Optical Density                                                         (O.D.) at 405 nm                                              ______________________________________                                        Negative Response:                                                                              <2 × Bkg.* at 1:40                                    Low Response:     >2 × Bkg. at 1:40, and                                                  ≦2 × Bkg. at 1:160                             Medium Response:  >2 × Bkg. at 1:40, and                                                  >2 × Bkg. at 1:160, but                                                 <4 × Bkg. at 1:640                                    High Response:    ≧4 × Bkg. at 1:640                             ______________________________________                                         *Bkg. = Background O.D. of samples not containing serum (i.e. serum           controls).                                                               

                  TABLE 4-1                                                       ______________________________________                                        ELISA Results of Toxoid Immunized Mice                                        Type of Response                                                                             % of Mice Tested                                               ______________________________________                                        Negative       12.5                                                           Low            6.25                                                           Medium         59.4                                                           High           21.8                                                           ______________________________________                                    

Based on these data, it is concluded that there was a total ofapproximately 87% antibody responders to vaccination with as little as a15 μg dosage.

EXAMPLE 5 Toxoid Stability

To demonstrate that the toxoid does not revert to the toxic state,toxoid (Ex. 1) and toxin (Prep. 1) were dialyzed exhaustively againstPBS for 1 week at RT. and then the toxoid was allowed to remain for anadditional three weeks at 4° C. Samples were removed and tested forenzyme activity and the following data were obtained.

                  TABLE 5-1                                                       ______________________________________                                                  Enzyme Activity (cpm/μg protein)                                 ______________________________________                                        Toxin       947                                                               Toxoid (Day 0)                                                                            90                                                                Toxoid (1 Wk.)                                                                            91                                                                Toxoid (1 Mo.)                                                                            92                                                                ______________________________________                                    

Based on these data, it is concluded that the toxoid remains stable anddoes not revert to toxin over at least a 1-month period.

Following the procedure of Example 1, toxoids may be prepared from otherADP-ribosylating toxins such as those from E. coli, Vibrio cholerae, andCorynebacterium diphtheriae.

The toxoids of this invention may be used in mammalian species foreither active or passive immunization prophylactically ortherapeutically against disease caused by the corresponding organism.Passive vaccination can be accomplished by injecting either wholeantiserum or immune globulin obtained from mammals previously vaccinatedwith the toxoid, with or without a pharmaceutically acceptable carrier.Such globulin is obtained by standard techniques from whole antiserum.

In a preferred embodiment of this invention, the exotoxoid of Example 1is used in combination with a vaccine, which combination offers muchbroader protection against gram-negative bacteremia. The secondcomponent is used to raise antibodies against gram-negative bacterialendotoxins. The preferred organisms are Salmonella minnesota Re 595 andthe J-5 mutant of E. coli 0111 B 4. These are preferred as they appearto raise antibodies against core glycolipids common to gram-negativeendotoxin and, therefore, offer a broader spectrum of protection thanorganisms which would merely raise antibodies specific to themselves.The use of J-5 is taught in Braude et al., Antiserum treatment ofgram-negative bacteremia, Schweiz. Med. Wschr. 108, No. 48, pp.1872-1876 (1978). An unrestricted permanent deposit of the J-5 E. coliorganism used herein was made with the American Type Culture Collectionon Jan. 21, 1982 under accession No. ATCC 39041. Although other E. colistrains may be used in the practice of this invention, ATCC 39041 ispreferred.

In the practice of this preferred embodiment, antisera are raised astaught by Braude et al. or by Ziegler et al., Trans. Assoc. of Amer.Phys., XCI, 253-258 (1978). These antisera are then combined with toxoidantisera to form a bivalent immuno-therapeutic or prophylactic.Alternatively, and preferably, the immuno-globulins of these antiseraare used instead of the whole antisera.

Therefore, the toxoids of this invention are used in injectable form foractive, prophylactic immunization of mammalian species against diseasecaused by the corresponding organism. Alternatively, immunoglobulinderived from said toxoids may be used for passive immunization,prophylactically or therapeutically. When P. aeruginosa toxoid is used,it is combined with antibodies raised against bacterial endotoxins. Whenthe P. aeruginosa toxoid is combined with gram-negative bacterialendotoxin vaccine or derivatives thereof such as antisera orimmunoglobulin, the injectable form offers much broader protectionagainst gram-negative bacteremia. By the injectable form of the toxoidsof this invention is meant an effective amount of said toxoids, antiseraderived from said toxoids, gammaglobulin or other antibody-containingfractions of said antisera, said toxoids, antisera, or fractions beingused singly or in combination with a gram-negative bacterial endotoxinvaccine, antisera obtained from said endotoxin vaccine, or gammaglobulin or other antibody-containing fractions of said antisera, saidinjectable form further optionally comprising a pharmaceuticallyacceptable carrier, such as aseptic saline water. The use of anacceptable adjuvant (e.g., alum) is also intended to be within the scopeof this invention. In non-human mammals, a complete or incompleteadjuvant (e.g., Freund's) can be used.

Although the toxoids of this invention have not been tested in humans,the mouse data of Example 4 suggest that at least 5-25 μg toxoid areeffective to induce an antibody response in mammals, i.e., that such anamount is an effective amount for immunization or to produce antisera involunteer subjects. For the production of antisera on a long term basis,booster injections at 2-week intervals may be necessary. Likewise, fromthe data of Example 6, it is calculated that at least about 90 ml ofhuman antiserum raised against bacterial endotoxins having a minimum PHA(passive hemagglutination assay) titer of 1:32 is required to protect a70 kg human (1.25 ml or greater per kg of body weight) againstgram-negative bacterial endotoxins.

Preparation 2 Mice Immunosuppression

The mice used in Example 4 were also used in Example 6. They wereimmunosuppressed after developing antibodies to toxin in order to obtaina model which better resembles a clinically relevant situation.

Caesarian-derived, barrier-sustained, outbred albino (CFl) mice fromCharles River were used. Mice were 5.5 to 7 weeks old (20 to 24 grams)at the time of challenge.

Mice were immunosuppressed one day before challenge withcyclophosphamide (CYTOXAN, Meade Johnson & Co.). The Cytoxan wasdisolved in sterile, pyrogen-free, distilled water at a concentration of20 mg/ml (Cytoxan also contains NaCl for isotonicity). The concentrationof the solution was adjusted with sterile, pyrogen-freephosphate-buffered saline to deliver by intraperitioneal injection theappropriate dose (400 mg/kg) in 1 ml.

Preparation 3 P. Aeruginosa Challenge

Frozen bacterial stock of a clinical isolate of P. aeruginosa was thawedthe day before the challenge, innoculated on a trypticase soy agarslant, and incubated at 37° C. overnight. The next day the bacteria weresuspended in 2.5 ml of phosphate-buffered saline inoculated into 100 mlof trypticase soy broth, and incubated at 37° C. in a shaker-incubator.When the bacteria reached mid-logarithmic growth, they were washed 3times in phosphate-buffered saline, and resuspended in PBS at aconcentration of 7×10⁶ bacteria/ml. The mice were given 0.1 ml of thesuspension intraperitoneally (.e., LD₉₅ dose).

EXAMPLE 6 Mice Immunization

In order to demonstrate the immunogenicity of toxoid, prepared as inExample 1, mice with antibody titer distributions listed in Table 4-1were immunosuppressed and challenged with P. aerugina by intraperitonealinjection (Prep. 3). Positive and negative controls were used as well astreatment with placebos. The improved efficacy achieved by combining thesaid toxoid active immunization and passive immunization with antiseraobtained from J-5 E. coli-vaccinated human volunteers was alsodemonstrated. The antisera were obtained following the proceduredescribed in Ziegler et al., Trans. Assoc. of Amer. Phys., XCI, 253-258(1978). The J-5 E. Coli organism used is now on deposit with theAmerican Type Culture Collection under Accession No. ATCC 39041. Thedata of Table 6-1 were obtained.

These data show that:

(1) Untreated, challenged animals die quickly (VI).

(2) Untreated, unchallenged animals die later of natural infections (V).

(3) J-5 antiserum alone offers some protection (III vs. VI).

(4) The combination of toxoid treatment and passive J-5 antiserumtreatment is efficacious (I vs. II).

(5) Because of 4, the toxoid is immunogenic.

(6) Combined treatment also protects against natural infections (I vs.V).

                                      TABLE 6-1                                   __________________________________________________________________________    Protection Against P. aeruginosa Infections                                   In Mouse Immunosuppression Model                                                           No. of Deaths       Day 5                                        Treatment    Day 1                                                                             Day 2                                                                             Day 3                                                                             Day 4                                                                             Day 5                                                                             Dead/Total                                                                          % Survival                             __________________________________________________________________________    I. Toxoid Immune.sup.A                                                                     0   1   0   2   0    3/32 91                                        + Passive Post.J-5.sup.B                                                   II.                                                                              Toxoid Immune                                                                           0   0   1   5   9   15/31 52                                        + Passive Pre-J-5                                                          III.                                                                             Placebo   0   0   1   3   10  14/33 58                                        + Passive Post-J-5                                                         IV.                                                                              Placebo   0   0   2   3   7   12/32 63                                        + Passive Pre-J-5                                                          V. No Treatment                                                                            0   0   0   2   10  12/39 69                                        + No Challenge                                                             VI.                                                                              No Treatment                                                                            25  10  2   0   0   37/39  5                                        + Challenge                                                                __________________________________________________________________________     .sup.A Animal immunized per Ex. 4; challenged on Day 29 post initial          immunization day.                                                             .sup.B Equivalent to 25 μl of human volunteer antiserum having a PHA       titer of 1:32 and given i.p. 4 hours prior to challenge.                 

What is claimed is:
 1. Toxoids which are ##STR4## wherein R is theADP-ribosylating toxin radical exotoxin-A from P. aeruginosa heat labileenterotoxin from E. coli, cholera enterotoxin from V. cholerae, ordiphtheria exotoxin from C. diphtheriae.
 2. 8-Adenylamino P. aeruginosaexotoxin-A.
 3. 8-Adenosylamino P. aeruginosa exotoxin-A.
 4. Acomposition comprising an antitoxin effective amount of the toxoid ofclaim 1, antiseria raised against said toxoid, gammaglobulin or otherantibody-containing fractions of said antisera, and a pharmaceuticallyacceptable carrier.
 5. The composition of claim 4 wherein the toxoidis(1) 8-adenylamino P. aeruginosa exotoxin-A or 8-adenosylamino P.aeruginosa exotoxin-A, and (2) antiserum raised against J-5 E. coli,ATCC 39041.